Estandarización de un protocolo sencillo para la extracción de ADN genómico de levaduras
<p class="MsoNormal" style="margin: 0cm 0cm 0pt; mso-layout-grid-align: none;"><strong><span style="font-size: 10pt; color: #4d4d4d; font-family: AGaramondPro-Bold; mso-bidi-font-family: AGaramondPro-Bold; mso-ansi-language: EN-US;" lang="EN-US"...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | Spanish |
| Published: |
Universidad Nacional de Colombia
2009-10-01
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| Series: | Revista Colombiana de Biotecnología |
| Subjects: | |
| Online Access: | http://www.revistas.unal.edu.co/index.php/biotecnologia/article/view/10339 |
| Summary: | <p class="MsoNormal" style="margin: 0cm 0cm 0pt; mso-layout-grid-align: none;"><strong><span style="font-size: 10pt; color: #4d4d4d; font-family: AGaramondPro-Bold; mso-bidi-font-family: AGaramondPro-Bold; mso-ansi-language: EN-US;" lang="EN-US"><span style="font-family: Times New Roman;">Standardising a simple protocol for extracting yeast from genomic DNA</span></span></strong></p><p class="MsoNormal" style="margin: 0cm 0cm 0pt; mso-layout-grid-align: none;"><strong><span style="font-size: 10pt; color: black; font-family: AGaramondPro-Bold; mso-bidi-font-family: AGaramondPro-Bold; mso-ansi-language: EN-US;" lang="EN-US"><span style="font-family: Times New Roman;"> </span></span></strong></p><p class="MsoNormal" style="margin: 0cm 0cm 0pt; mso-layout-grid-align: none;"><strong><span style="font-size: 10pt; color: black; font-family: AGaramondPro-Bold; mso-bidi-font-family: AGaramondPro-Bold;"><span style="font-family: Times New Roman;">Resumen: </span></span></strong><span style="font-size: 10pt; color: black; font-family: Garamond; mso-bidi-font-family: Garamond;">Se estandarizó un protocolo rápido, sencillo y de bajo costo para la extracción de ADN genómico de levaduras a partir de lisis de la pared celular mediante tratamiento enzimático y precipitación por alcoholes. El empleo de la enzima Beta-glucoronidasa en reemplazo de la enzima Zimolasa, permitió obtener ADN en alta concentración (124,9±30,2 ng/μl) y de buena calidad (A260/A280 nm =1,86±0,1), ideal para su uso en estudios de biología molecular. Además, se adicionó un paso de incubación del ADN obtenido a 100° C para inactivar ADNasas. La calidad del ADN obtenido fue evaluada por medio de la amplificación de la región ITS1-5.8S-ITS2, presentando bandas definidas y cuantificables (entre 380 y 880 pb) ideales para estudios de identificación molecular y filogenia.</span></p><p class="MsoNormal" style="margin: 0cm 0cm 0pt; mso-layout-grid-align: none;"><strong><span style="font-size: 10pt; color: black; font-family: Garamond-Bold; mso-bidi-font-family: Garamond-Bold;"><span style="font-family: Times New Roman;">Palabras clave: </span></span></strong><span style="font-size: 10pt; color: black; font-family: Garamond; mso-bidi-font-family: Garamond;">levadura; extracción de ADN; Beta-Glucoronidasa.</span></p><p class="MsoNormal" style="margin: 0cm 0cm 0pt; mso-layout-grid-align: none;"> </p><p class="MsoNormal" style="margin: 0cm 0cm 0pt; mso-layout-grid-align: none;"><strong><span style="font-size: 10pt; font-family: AGaramondPro-Bold; mso-bidi-font-family: AGaramondPro-Bold; mso-ansi-language: EN-US;" lang="EN-US"><span style="font-family: Times New Roman;">Abstract: </span></span></strong><span style="font-size: 10pt; color: black; font-family: Garamond; mso-bidi-font-family: Garamond;">A quick, simple and low-cost protocol for extracting genomic DNA from yeast by cell wall lysis involving enzymatic treatment and alcoholic precipitation was standardised. Higher DNA yields (124.9±30.2 ng/μl) were obtained by using beta-glucuronidase instead of zymolyase; these had very high quality (A260/A280 nm = 1.86±0.1) and would be suitable for use in molecular biology assays. Moreover, a DNAse inactivation step was also introduced by incubation at 100 °C to further ensure DNA stability. </span><span style="font-size: 10pt; color: black; font-family: Garamond; mso-bidi-font-family: Garamond; mso-ansi-language: EN-US;" lang="EN-US">DNA quality was assayed by PCR amplification </span><span style="font-size: 10pt; font-family: Garamond; mso-bidi-font-family: Garamond; mso-ansi-language: EN-US;" lang="EN-US">of the ITS1-5.8S-ITS2 region, revealing defined, quantifiable 380 to 880 bp bands. These results show that the protocol is ideal for molecular identification and phylogenetic studies.</span><span style="font-size: 10pt; color: black; font-family: Garamond; mso-bidi-font-family: Garamond;"></span></p><p class="MsoNormal" style="margin: 0cm 0cm 0pt; mso-layout-grid-align: none;"><strong><span style="font-size: 10pt; font-family: Garamond-Bold; mso-bidi-font-family: Garamond-Bold; mso-ansi-language: EN-US;" lang="EN-US"><span style="font-family: Times New Roman;">Key words: </span></span></strong><span style="font-size: 10pt; font-family: Garamond; mso-bidi-font-family: Garamond; mso-ansi-language: EN-US;" lang="EN-US">Yeast; DNA extraction; beta-glucoronidase.</span></p> |
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| ISSN: | 0123-3475 1909-8758 |